Gubi decoction mitigates knee osteoarthritis via promoting chondrocyte autophagy through METTL3‐mediated ATG7 m6A methylation

Abstract Knee osteoarthritis (KOA) is a chronic joint disease that significantly affects the health of the elderly. As an herbal remedy, Gubi decoction (GBD) has been traditionally used for the treatment of osteoarthritis‐related syndromes. However, the anti‐KOA efficacy and mechanism of GBD remain unclear. This study aimed to experimentally investigate the anti‐KOA efficacy and the underlying mechanism of GBD. The medial meniscus (DMM) mice model and IL‐1β‐stimulated chondrocytes were, respectively, constructed as in vivo and in vitro models of KOA to evaluate the osteoprotective effect and molecular mechanism of GBD. The UPLC–MS/MS analysis showed that GBD mainly contained pinoresinol diglucoside, rehmannioside D, hesperidin, liquiritin, baohuoside I, glycyrrhizic acid, kaempferol and tangeretin. Animal experiment showed that GBD could alleviate articular cartilage destruction and recover histopathological alterations in DMM mice. In addition, GBD inhibited chondrocyte apoptosis and restored DMM‐induced dysregulated autophagy evidenced by the upregulation of ATG7 and LC3 II/LC3 I but decreased P62 level. Mechanistically, METTL3‐mediated m6A modification decreased the expression of ATG7 in DMM mice, as it could be significantly attenuated by GBD. METTL3 overexpression significantly counteracted the protective effect of GBD on chondrocyte autophagy. Further research showed that GBD promoted proteasome‐mediated ubiquitination degradation of METLL3. Our findings suggest that GBD could act as a protective agent against KOA. The protective effect of GBD may result from its promotion on chondrocyte autophagy by suppressing METTL3‐dependent ATG7 m6A methylation.


| INTRODUC TI ON
Osteoarthritis (OA) is a common and degenerative joint disease characterized by the progressive degradation and destruction of articular cartilage.Generally, the knee is considered to be the most prevalent joint affected by osteoarthritis.Knee osteoarthritis (KOA) could lead to functional disability and affect nearly 40% of the aging population worldwide, imposing massive social and economic burdens. 1 To date, effective treatments for patients with KOA are still insufficient or poorly tolerated.Non-steroid anti-inflammatory drugs (NSAIDs) and some anti-KOA drugs, like diacerein and glucosamine, are only palliative for patients with mild and early KOA. 2,3For patients with advanced KOA, arthroplasty remains the only choice. 4The pathogenesis of KOA is multifactorial, and the risk factors mainly include genetics, previous trauma, age, obesity and stress, as could provoke articular cartilage degeneration. 5Recently, numerous evidence has demonstrated that chondrocyte apoptosis triggers the destruction of cartilage. 6,7However, autophagy, a highly conserved degradation system, exhibits a protective effect on damaged chondrocytes and alleviates the progression of KOA. 8 Actually, inhibiting the apoptosis of chondrocytes by autophagy activation has attracted much attention of scholar.
Autophagy is an important metabolic pathway to modulate inflammation and maintain stability of intracellular environment in the body. 9The impairment of autophagic activity could down-regulate clearance efficiency, eventually facilitate cell degeneration or even apoptosis. 10Recent studies have shown that, under the pathogenic conditions of KOA, the level of autophagy in chondrocytes decreases and is closely associated with the increased severity of KOA. 11Lian et al. reported that microRNA-128a repressed chondrocyte autophagy and exacerbates KOA by disrupting ATG12. 12tivation of autophagy could protect chondrocytes from degradation and apoptosis, and thus effectively alleviate the development of KOA.For example, restoring autophagy by mechanical loading or targeting TNFR1-associated death domain protein were proved to effectively retard OA pathological progression and mitigate OA symptoms at both early and late stages. 13,14Rapamycin, a selective mTOR inhibitor, was also reported to protect chondrocytes against IL-18-induced apoptosis and ameliorate OA via promoting autophagy. 15These findings suggest that activating autophagy of articular chondrocytes may be a promising strategy for KOA prevention.
In clinical practice, due to the advantages of remarkable curative effect, minimal side effects and low cost, herbal medicine has presented tremendous promise in OA treatment when compared to Western medicine. 16 (GU) (The plant names have been checked with http:// www.thepl antli st.org/ ). 17GBD has been traditionally applied in clinical practice to treat osteoporosis, KOA and other ageing diseases for several decades. 18Modern pharmacological studies showed that the effective components in GBD obviously protected against OA.For example, catalpol could prevent chondrocyte apoptotic level triggered by IL-1ß, and inhibit cartilage degeneration in the knee joint of OA rat. 19reover, Achyranthes bidentata polysaccharides, the active component of Achyranthis Bidentatae Radix, was proved to promote chondrocyte proliferation by activating the Wnt/β-catenin signalling pathway. 20Clinical investigations showed that GBD can effectively relieve pain and improve knee joint function in patients with OA, and has a significant inhibitory effect on serum TNFα and ILβ level. 21However, the specific mechanism that GBD against KOA is still unclear and remains to be fully elucidated.In the present study, we aimed to investigate the molecular mechanism underlying GBD in treating KOA.We constructed the mice model of KOA and cultivated IL-1β-induced chondrocytes to explore whether the protective effects of GBD was mediated by inhibiting chondrocyte autophagy.
On the morning of the 4th day, the abdominal aortic blood were collected and then centrifuged at 3000 rpm for 15 min.The plasma was inactivated by water bath (56°C, 30 min), filtered using 0.22 μm membrane, and stored at −20°C for in vitro study.

| Histological evaluation
At the end of the experiment, the tibia and femur tissues of mice were separated for general histological evaluation, including H&E staining and Safranin-O staining.The OARSI score was applied to estimate the severity of KOA.

| Quantitative micro-CT evaluation
In order to examine the effects of GBD on bone remodelling, quantitative evaluations of the tibial subchondral bone was performed using micro-CT (μCT) equipment (PerkinElmer).Micro-CT scans were performed with the following instrument settings: x-ray voltage, 90 kV; tube current, 88 μA; voxel size, 90 μm; exposure time, 14 min; continuous (non-stepping) rotation.

| Immunohistochemical (IHC) staining
The cartilage sections of knee joint were incubated with primary antibodies against LC3 at 4°C for overnight, and then incubated with goat anti-mouse/rabbit HRP-conjugated secondary antibody.
Subsequently, sections were processed with 3,3'-diaminobenzidine horseradish peroxidase colour development kit (Beyotime, Nanjing, China) prior to counterstaining with haematoxylin.Positively stained cells were visualized using light microscope and quantified using ImageJ software.

| Immunofluorescence (IF) staining
For IF staining, the slides from each treatment group were blocked with 5% BSA and then incubated with primary antibodies against MMP13 (Abcam, Cambridge, UK), LC3 (Abcam, Cambridge, UK) and ATG7 (Invitrogen, Carlsbad, USA) overnight at 4°C.The next day, the slices were incubated with a fluorescein-conjugated secondary antibody (Proteintech, Wuhan, China), and then the nuclei were stained with DAPI (Santa Cruz, USA).A fluorescence microscope (Zeiss, Germany) was used to acquire the images.

| TdT-mediated dUTP Nick-End Labelling (TUNEL)
The TUNEL Apoptosis Detection Kit (Abbkine Scientific Co., Ltd., Wuhan, China) was applied to evaluate chondrocyte apoptosis in sections.The slides were fixed and permeabilized with 0.1% Triton X-100, and then incubated with reagent mixture.The following day, the nuclei were stained with DAPI (Santa Cruz, USA), and then observed under an inverted fluorescence microscope (Olympus, Tokyo, Japan).

| Chondrocytes isolation and culture
Six-week-old SD rats were killed to harvest chondrocytes.The cartilage from both knees and hips were collected under sterile conditions.After that, the cartilage were cut into small pieces, washed and digested with 0.25% trypsin-EDTA solution and 0.2% type II collagenase.Next, the released chondrocytes were harvested and passaged when they reached 80%-90% confluency.Cells at passage 2 and 3 were used for the experiments.

| Cell viability assay
The methyl thiazol tetrazolium (MTT) assay was used to determine cell viability following IL-1β (10 ng/mL) and GBD treatment.Briefly, rat chondrocytes (1 × 10 4 /well) were seeded in 96-well plates for 24 h.The cells were then treated with IL-1β or GBD-containing serum, MTT assay was performed routinely.The optical density value was measured with a microplate reader at 570 nm.

| qRT-PCR and western blot
Total RNA and whole protein extraction of cartilage tissue or chondrocytes were respectively prepared for RT-PCR and western blot analysis.The primer sequences for qRT-PCR in this study were as follows: rat ATG7, forward: 5'-GAGAGCCGATGGCTTCCTAC-3', reverse: 5'-CCTGGA GCCACCACATCA TT-3'; mouse ATG7, forward:

| Flow cytometry analysis
Chondrocytes were seeded onto glass coverslips for overnight.
The cells were then stimulated with IL-1β (10 ng/mL) for 1 h following GBD treatment for 24 h.After that, apoptosis analysis was conducted using Annexin V-FITC/PI Apoptosis Detection Kit (Keygen Biotech, Jiangsu, China) according to the manufacturer's instructions.The data were detected with a flow cytometry.

| m 6 A quantification
The global m 6 A levels of mRNA were determined with an m 6 A RNA Methylation Assay Kit (Colorimetric) following the manufacture's protocol.The absorbance was measured at 450 nm, and the total m 6 A content was calculated based on the standard curve.

| MeRIP-qPCR
Total RNA was obtained and fragmented.A part of the fragmented RNA was saved as input, and the rest RNA sample was immunoprecipitated with beads conjugated by m 6 A-antibody, and then eluted.The m 6 A immunoprecipitated samples were further analysed using qPCR.The relative enrichment of m 6 A was normalized to the input.
Total RNAs were isolated, and the half-life of mRNA was detected by qPCR.

| METTL3 overexpression
To METTL3 overexpression, lentivirus containing METTL3overexpressing fragment were transfected into chondrocytes using Lipofectamine3000 (Invitrogen).After transfection for 24 h, cells were harvested for subsequent experimental measurement.The scramble shRNA was transfected as a negative control.

| Statistical analyses
All data are presented as the means ± SEM.A Student's t-test (twotails) was applied for comparison between two groups, while oneway anova was employed for comparisons involving multiple groups.
Differences were considered statistically significant when p < 0.05.

| Identification of the compounds in GBD extract by LC-MS/MS
Firstly, the major chemical ingredients GBD extract were characterized using LC-MS/MS.The representative total ion chromatograms of standard compounds and GBD extract in negative ion mode were shown as Figure 1A,B.According to measured molecular weight, fragment ion, elemental analysis, and compared with standard chemicals, eight compounds in GBD extract were definitely identified to be pinoresinol diglucoside (peak 1), rehmannioside D (peak 2), hesperidin (peak 3), liquiritin (peak 4), baohuoside I (peak 5), glycyrrhizic acid (peak 6), kaempferol (peak 7) and tangeretin (peak 8).

| GBD effectively prevented progression of KOA in DMM mice
To determine the efficacy of GBD on development of KOA, the changes in microstructure of the subchondral bone was evaluated by 3-D reconstruction of subchondral bone using quantitative micro-CT.
The results showed that GBD could protect the subchondral bone of DMM mice in a dose-dependent manner (Figure 2A).GBD-treated group showed decreased knee joint diameter, increased bone volume and bone surface when compared with the model group (Figure 2B-D).
To elevate the therapeutic effect of GBD on cartilage damage in KOA, the joint of mice was excised, and H&E staining as well as Safranin-O fast green staining were conducted.As seen in Figure 2E, mice treated with GBD exhibited decreased width and proteoglycan of cartilage component in comparison to model group.Furthermore, GBD decreased cartilage degradation of the knee joint.Consistent with the observed histopathological alterations in the cartilage of mice, GBD significantly reduced cartilage OARSI score elevated by DMM (Figure 2F).
Moreover, GBD inhibited the mRNA levels of inflammatory cytokines including TNFα, IL6, and IL-1β in articular cartilage (Figure 2G).Together, these experimental results indicated that GBD could effectively prevented progression of KOA by reducing bone and cartilage damage.

| GBD ameliorated cartilage degeneration and inhibited chondrocyte apoptosis
Articular cartilage is a connective tissue that mainly composed of chondrocytes and extracellular matrix (ECM).We demonstrated the protective effect of GBD on cartilage destruction in DMM mice.
Next, we further examined whether GBD affected ECM degradation and chondrocytes apoptosis.Cartilage synthesis metabolism indicators such as collagen II, Runx2, SOX9 and MMP13 were determined by qRT-PCR.It was found that GBD could promote the mRNA expression of collagen II, Runx2 and SOX9, but inhibited the mRNA level of MMP13 (Figure 3A).MMP-13 is a major metalloproteinase that involved in the cleavage of type II collagen and cartilage degradation.In our study, different methods were applied to detect the expression of MMP-13.Results from IF staining and western blot analysis both showed that high expression of MMP13 was induced after DMM surgery (Figure 3B,C).Treatment with GBD markedly reduced MMP13 levels in the cartilage tissue of DMM mice (Figure 3B,C).Additionally, TUNEL staining (in green) were conducted to assess the degree of chondrocyte apoptosis.We found that the number of TUNEL-positive cells significantly increased in the DMM mice but reduced after GBD treatment (Figure 3D,E).

| GBD restored DMM-induced dysregulated autophagy in cartilage tissue of mice
3][24] Autophagy deficiency would exacerbate cell apoptosis.Therefore, we investigated the regulation effect of GBD on chondrocyte autophagy in this process.As expected, by applying immunohisto-

| GBD repaired chondrocyte autophagy by suppressing ATG7 m 6 A modification
ATG7 is an autophagy effector enzyme that regulate the cell apoptosis.N6-methyladenosine (m 6 A) is an important and reversible RNA methylation.A recent evidence has suggest that Mettl3-mediated m 6 A could control autophagy by targeting ATG7 and then accelerate the progression of KOA. 25 Since GBD enhanced the expression of ATG7 and restored DMM-induced dysregulated autophagy, we wonder whether METTL3-mediated m 6 A is involved in the regulation of GBD on ATG7 expression.The m6A level of RNA was determined by using an m6A RNA methylation assay kit, and METTL3 expression was detected using western blot.The results indicated that the level of m6A in the DMM mice was much higher than in the sham group (Figure 5A), and the level of METTL3 expression also profoundly increased (Figure 5B,C).GBD could markedly inhibit the level of m6A as well as METTL3 expression.To verify the role of METTL3-mediated m 6 A modification in chondrocyte autophagy, we further examined the mRNA levels of ULK1, ATG13, Beclin1, ATG5, ATG12 and ATG7 by QPCR (Figure 5D-I).Consistent with the ATG7 protein level, ATG7 mRNA were significantly reduced by DMM surgery, and upregulated after GBD treatment (Figure 5I).Furthermore, MeRIP-qPCR indicated that DMM augmented the m 6 A modification of ATG7 in the model group, whereas GBD administration attenuated the level (Figure 5J).Together, METTL3-mediated ATG7 m 6 A modification might contribute to the enhancement of GBD on chondrocyte autophagy.

| GBD inhibited chondrocyte apoptosis and attenuated autophagy deficiency in vitro
The chondroprotective effects of GBD were further instigated in However, the cells treated GBD showed increased incidence of autophagy compared with the IL-1β-stimulated cells (p < 0.01).To determine whether the protective effect of GBD on chondrocyte is due to its activation of autophagy, we examined the effects of pharmacological inhibition by 50 nM chloroquine on GBD-mediated beneficial regulation on chondrocyte apoptosis.The results showed that autophagy inhibition by chloroquine significantly reduced the anti-apoptotic effect of GBD in chondrocyte cells (Figure S1).

| GBD exerted chondroprotective effect by inhibiting METTL3-mediated ATG7 m 6 A modification in vitro
Finally, we investigated whether GBD exerted chondroprotective effects by influencing ATG7 m 6 A modification in chondrocyte cells.
Consistent with the animal study, GBD treatment decreased the level of m 6 A and METTL3 expression (Figure 7A-C).GBD could also effectively reverse the decline of ATG7 mRNA as well as half-life changes induced by IL-1β (Figure 7D-F).Furthermore, we found that GBD significantly inhibited IL-1β-induced increase of m 6 A levels of ATG7 in chondrocyte cells, based on the MeRIP-qPCR analysis (Figure 7G).To assess whether ATG7 m 6 A modification were required for chondroprotective effect of GBD, we constructed METTL3 mutant recombination plasmid for METTL3 overexpression (Figure 8A).Our data showed that METTL3 overexpression had little effect on the LC3II/ LC3I ratio and ATG7 level when compared with the empty vector (Figure 8B-D).Nevertheless, overexpression of METTL3 significantly counteracted the enhanced autophagy as well as chondrocyte apoptosis inhibition (Figure 8E,F) induced by GBD, implying that GBD regulated chondrocyte autophagy and apoptosis through targeting ATG7 m 6 A modification in a METTL3-dependent manner.

| GBD promoted proteasome-mediated ubiquitination degradation of METLL3
It is well known that proteasome-dependent and lysosomedependent degradation are two important ways of protein degradation.In order to explore how GBD regulates the progression of OA, we sought to investigate the mechanism by which GBD regulates METLL3 degradation.Coincubation of specific lysosomal inhibitor cycloheximide displayed little effect on METLL3 downregulation (Figure 8G), whereas the specific proteasome inhibitor MG132 abolished the degradation of METLL3 by GBD (Figure 8H).
Consistently, we also found that the METLL3 ubiquitination was markedly increased in the presence of GBD (Figure 8I).

| DISCUSS ION
KOA is a common joint disease that imposes huge social costs on society.The current conventional treatment for KOA such as NSAIDs could not directly protect cartilage against injuries, and cause kinds of adverse effects.Therefore, the use of natural ways, including herbal formulas, has garnered much interest for its effectiveness on KOA and absence of side effects. 26Herbal formula GBD has been widely used to ameliorate KOA progression, but has not been adequately studied.In this work, the anti-osteoarthritic effect of GBD and its underlying mechanism were investigated.Osteoarthritis is characterized by articular cartilage degradation, subchondral osteosclerosis, osteophyte formation, and synovitis. 27The DMM mouse model is a common in vivo model to mimic the degenerative process during KOA. 28IL-1β, as an inducer of OA microenvironment, has been recognized as a homogenous substance for establishing cellular KOA models. 29In the present study, both DMM-induced KOA Generally, KOA is characterized by progressive cartilage degradation, which is mainly caused by chondrocyte apoptosis or cytokine production. 30Cartilage is an integral part of the skeletal system, and contains chondrocytes which produce the ECM required for cartilage homeostasis. 31Proper cartilage development is essential to bone formation.The morphological and molecular features of chondrocyte apoptosis, including loss of nuclear volume and apoptotic bodies, have been detected in human KOA tissue specimens, and positively correlated with the severity of cartilage destruction. 17Currently, the apoptotic mechanisms in cartilage degeneration in KOA have been thoroughly explored. 32,33It has been widely reported that transforming growth factorβ(TGF-β), 34 fibroblast growth factors (FGFs), 35 mitogen-activated protein kinase, and Hippo/YAP signalling 36 tightly modulate the cellular processes of chondrocytes.NO has been previously demonstrated to be the main player in chondrocyte apoptosis.In the presence of NO, a low level of ROS would induce chondrocyte apoptosis while high concentrations of ROS induce necrosis. 37Incubation of human articular chondrocytes with sodium nitroprusside (a chemical NO donor) increased gene expression of caspase-3, caspase-7, and downregulated bcl-2 mRNA levels. 38On the other hand, chondrocyte apoptosis could also be induced in caspase-independent manner, involving peroxynitrite-induced mitochondrial dysfunction, calpains as well as calcium-dependent cysteine proteases. 39Given the antidegenerative effects of GBD against KOA, we further demonstrated whether GBD inhibited chondrocyte apoptosis in articular cartilage.In animal study, TUNEL staining was conducted to assess the degree of chondrocyte apoptosis.We found that, the number of TUNELpositive cells significantly increased in the DMM mice but reduced after GBD treatment (Figure 3D,E).Furthermore, in IL-1β-stimulated chondrocytes, GBD treatment could remarkably reduce the cell apoptosis and increase cell viability, suggesting that GBD might produce chondroprotective effect through inhibiting chondrocyte apoptosis.
Next, we further investigated the action of GBD in regulating chondrocyte apoptosis.Our observation indicated that GBD may play an anti-apoptotic role in chondrocytes by regulating autophagy.
Autophagy is a conserved survival mechanism that protects cells, including chondrocytes, from apoptosis. 21During autophagy, cytosolic LC3-I is conjugated to phosphatidylethanolamine and converted to LC3-II. 40The ratio of LC3-II/LC3-I is widely considered as an indication of autophagy activation.Besides, autophagy is tightly regulated by autophagy-related genes (ATGs) such as ATG7, LC3 and P62.P62 is an autophagy adaptor protein that recognizes unwanted cellular wastes, ATG7 coordinate the phagophore elongation, and Beclin1 is a core regulator of autophagosome biogenesis. 41Indeed, dysregulated autophagic activity has been widely reported during KOA development, and promotion of autophagic flux has been recognized as a promising therapeutic strategy for KOA. 42,43Previous reports have shown that the protein and mRNA levels of LC3-II, an autophagy marker, were decreased in articular cartilage of OA patients and in an animal OA model. 44,45Activation of autophagy via ERK1/2 signalling pathways could inhibit apoptosis of chondrocytes. 46In this study, we assessed autophagy by measuring the protein abundance of LC3, P62 and ATG7 as well as the ratio of LC3-II/LC3-I.We found that autophagy was significantly inhibited in DMM mice and chondrocytes under the stimulation of IL-1β (Figure 6D-H).GBD treatment effectively promoted the autophagic activity as evidenced by increased ATG7 and LC3-II expression.Furthermore, inhibiting autophagy using chloroquine significantly reduced the anti-apoptotic effect of GBD in chondrocytes (Figure S1), confirming that GBD protected chondrocyte form apoptosis by strengthening autophagy.Since tt has been reported that improving autophagy can effectively alleviate chondrocyte apoptosis and osteoarthritis, 14 we speculate that GBD may play an anti-KOA role by promoting autophagy.
Emerging as an abundant RNA modification in eukaryotic mRNA, m 6 A regulates a variety of biological processes such as gene expression, cell fate, and involves pathological and physiological events.m 6 A is triggered by methyltransferase ('writers', e.g., METTL3, METTL14), and removed by demethylase ('erasers', e.g., FTO, ALKBH5). 47m 6 A modifications affect the stability, selective splicing and translation of RNA, and thus affect apoptosis, autophagy and immune response. 48Recent studies found that disorder of m 6 A contributes to pathological bone diseases including KOA. 49,50 In addition to this, a recent work has reported that METTL3-mediated m 6 A modification of ATG7 regulates autophagy to promote the OA progression. 25Given the promotion of GBD on ATG7 expression and chondrocyte autophagy, we further examined its regulation on m 6 A level of ATG7 and METTL3 expression.Our results m 6 A modification of ATG7 was significantly elevated in the model group, accompanied by increased METTL3 level.GBD effectively suppressed ATG7 m 6 A modification and METTL3 expression in vivo and in vitro studies (Figures 5 and 7).
chemistry and IF analysis, DMM surgery significantly inhibited the expression of autophagy markers including LC3II (Figure 4A,B) and Atg7 (Figure 4C,D).Moreover, western blot analysis also confirmed the degradation of autophagy markers in model group.GBD treatment could enhance the expression of Atg7 and LC3 II and decreased P62 levels in DMM mice (Figure 4E-H).These results indicated that the antiapoptotic activity of GBD in chondrocyte might be associated with its positive regulation of autophagy.F I G U R E 1 Representative LC-MS/MS chromatograms of GBD in negative ion mode.(A) Standard compounds.(B) GBD extract.

F I G U R E 2 | 7 of 14 CUI
The protective effect of GBD against DMM-induced knee osteoarthritis.(A) Representative micro-CT images of subchondral bone of mice.(B-D) Knee joint diameter, bone volume and bone surface of mice from different groups.(E) H&E staining and safranin-O/fast green staining of cartilage areas.(F) OARSI score.(G) TNFα, IL-1β and IL-6 mRNA levels in articular cartilage.## p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus model group.et al.
isolated rat chondrocytes in vitro.MTT assay and Annexin-V/PI flow cytometry results showed GBD treatment could significantly improve cell viability and inhibited IL-1β (10 ng/mL)-induced chondrocyte apoptosis (Figure 6A,B).Next, IF staining and Western blot suggested that the addition of IL-1β significantly reduced the autophagy proteins LC3, ATG7, and the LC3II/LC3I ratio, accompanied by enhanced expression of p62 in chondrocyte cells (Figure 6D-H).

F I G U R E 3
GBD ameliorated cartilage degeneration and inhibited chondrocyte apoptosis.(A) The mRNA expression of collagen II, Runx2 and SOX9 in articular cartilage.(B) Immunohistochemistry assay of MMP13 protein.(C) Western blot images and quantitative analysis of MMP13 in cartilage tissue.(D) Apoptotic cells in cartilage tissue.(E) Graph shows the number of TUNEL-positive cells.## p < 0.01 versus control group; *p < 0.05, **p < 0.01versus model group.

F I G U R E 4
GBD restored DMM-induced dysregulated autophagy in cartilage tissue of mice.(A, B) Immunohistochemistry and quantification of LC3 positive cells.(C, D) Immunohistochemistry assay of ATG7 protein.(E) Western blot images of ATG7, LC3I, LC3II and P62 in cartilage tissue.(F-H) Quantitative analysis of western blot assay in indicated groups.## p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus model group.
mice model and IL-1β-induced cellular model were established.The results showed that GBD could inhibit glycosaminoglycan loss and cartilage destruction, decreased the OARSI scores.The efficacy of high doses of GBD (34.96 g/kg/day) is close to that of positive drug diclofenac (10 mg/kg/day).Additionally, GBD increased protein expressions of ATG7 and LC3II, whereas decreased P62 levels via suppressing ATG7 m 6 A modification.GBD also restored the IL-1βinduced chondrocyte apoptosis and autophagy deficiency in vitro.METTL3 overexpression significantly counteracted the protective effect of GBD on chondrocyte autophagy.These findings highlight the anti-degenerative effects of GBD against KOA, as might be mediated by promoting chondrocyte autophagy via METTL3-dependent ATG7 m 6 A methylation.

F I G U R E 5
GBD suppressed ATG7 m 6 A modification in vivo.(A) The m 6 A level of RNA in each group (n = 3).(B) Western blot image of METTL3 in cartilage tissue.(C) Quantitative analysis of western blot assay in indicated groups.(D-I) ULK1, ATG13, Beclin1, ATG5, ATG12 and ATG7 mRNA detected by qPCR.(J) The effect of GBD on the m 6 A modification of ATG7 was detected by MeRIP-PCR.## p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus model group.

F I G U R E 6 | 11 of 14 CUI
GBD inhibited chondrocyte apoptosis and attenuated autophagy deficiency in IL-1β-induced chondrocytes.(A) Cell viability evaluated by MTT.(B, C) Chondrocyte apoptosis detected by flow cytometry.(D, E) Representative images and quantification of GFP-LC3 in indicated groups.(F) Representative western blot of ATG7 and quantitative analysis data.(G) Representative western blot of LC3I, LC3II and quantitative analysis data.(H) Representative western blot of P62 and quantitative analysis data.## p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus model group.et al.

F I G U R E 7
GBD inhibited METTL3-mediated ATG7 m 6 A modification in vitro.(A) The m 6 A level of RNA in each group.(B) Western blot image of METTL3 in rat chondrocytes.(C) Quantitative analysis of western blot assay in indicated groups.(D) ATG7 mRNA detected by qPCR.(E) The effect of GBD on the RNA stability of ATG7.(F) The effect of GBD on the half-life of ATG7 mRNA.(G) m 6 A modification of ATG7 detected by MeRIP-PCR.## p < 0.01 versus control group; *p < 0.05, **p < 0.01 versus model group.
RG, EU, EB, AB, CR, BO, CT and GU were purchased from Tong Ren Tang (Hanzhou, China).Voucher specimens of the above herbs were stored in the herbarium of Zhejiang Chinese Medical lows: RG 15 g, EU 15 g, EB 15 g, AB 15 g, CR 15 g, BO 10 g, CT 10 g and GU 9 g, were mixed.Crude drugs were boiled in 10 volume (v/w) of distilled water for 1 h.Following filtration, the residue was further extracted in 8 volume of water for 1 h.Combined filtrate was concentrated under reduced pressure to 1 g crude drug/mL.